XMRV not detected in UK chronic fatigue syndrome patients

7 January 2010

A new retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), first identified in tumor tissue of individuals with prostate cancer, was subsequently found in 68 of 101 US patients with chronic fatigue syndrome (CFS). This observation raised the possibility that XMRV is the etiologic agent of CFS. An important question is whether XMRV is associated with CFS in other parts of the world. For some CFS patients in the UK the answer appears to be no.

The subjects of this study were 186 confirmed CFS patients who had been referred to the CFS clinic at King’s College Hospital, London. DNA was prepared from blood samples and subjected to polymerase chain reaction using primers that anneal to an XMRV-specific sequence, and to a sequence conserved among murine leukemia viruses. To demonstrate that the amplification worked, a positive control reaction was done using XMRV DNA. The reaction products were fractionated by electrophoresis on an agarose gel, and the DNAs were visualized by staining with a dye. As shown in the figure, positive control samples containing XMRV DNA produced DNAs of the expected sizes (lanes 10, 11). None of the 186 test DNAs from CFS patients produced a PCR product with either the XMRV or murine leukemia virus primers. Some of these negative samples are shown in lanes 1-8 of the figure.

The authors of this study suggest that they were more careful at avoiding contamination than the group which previously identified XMRV in American CFS patients:

…the PCR operator was blinded to the provenance of the DNA samples. In fact, with the exception of the PCR controls, all 186 DNA test samples originated from CFS patients. Care was taken to grow the XMRV plasmid in a laboratory in which no MLV had been cultured and no MLV vectors used and the PCR was carried out in a CPA-accredited Molecular Diagnostics Unit which processes only human tissue. Multiple (six) water (negative) controls were included in every run to detect low level contamination and a PCR to amplify a sequence that is conserved in most murine leukaemia viruses was included in order to expose any circulating MLV contamination and to detect any variant of XMRV that might be circulating in the UK CFS population.

But they also acknowledge that there might be population differences in XMRV distribution:

Based on our molecular data, we do not share the conviction that XMRV may be a contributory factor in the pathogenesis of CFS, at least in the U.K.

These results are surely a disappointment to CFS sufferers who believe that XMRV is the etiologic agent of the disease. But they reveal the dangers of making conclusions about disease etiology based on the findings of limited studies. As I wrote previously, while the presence of XMRV in 67% of CFS samples seems impressive, it could be misleading. For example, the samples could be from regions where XMRV infection is common.

In light of these new findings, it is informative to recall that the nucleic acid of another retrovirus, human T-lymphotropic virus type ll, was previously amplified from blood cells of 23 of 30 CFS patients, but not from healthy patients. However this observation was not confirmed in subsequent studies of CFS patients. In addition, there is no evidence for the presence of other retroviruses in CFS patients, including HIV-1, bovine and feline leukemia viruses, simian T lymphotropic virus type l, foamy virus, and simian retrovirus.

It is possible that XMRV is involved in CFS but only in certain parts of the world. More extensive studies such as the one reported here must be done worldwide to clarify the role of XMRV in CFS, and to determine whether other infectious agents are involved.

Official Statement from the Whittemore Peterson Institute Regarding UK Study

The Whittemore Peterson Institute (WPI) has reviewed the paper entitled “Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome.” This study did not duplicate the rigorous scientific techniques used by WPI, the National Cancer Institute and the Cleveland Clinic, therefore it cannot be considered a replication study nor can the results claim to be anything other than a failure not just to detect XMRV, but also a failure to suggest meaningful results.

The scientific methods used by WPI are very exact and require specific techniques to ensure accuracy. Differences in techniques employed by Erlwein et al. not only explain their failure to replicate the WPI study, but also render the conclusions meaningless. These differences include, but are not limited to the following:

1) blood sample volumes and processing; 2) patient criteria/population differences; 3) number and type of tests done to assure accurate results, including white blood cell culture; 4) use of a molecular plasmid control in water versus a positive blood sample; and 5) different primer sequences and amplification protocol used to find the virus, which were not validated by a clinical control.

The WPI study was published after six months of rigorous review and three independent lab confirmations, proving that contamination had not taken place and that infectious XMRV was present in 67 percent of CFS patients diagnosed according to the Canadian and Fukuda criteria. In contrast, this latest study was published online after only three days of review. Significant and critical questions remain as to the status of patient samples used in the UK study as those samples may have been confused with fatigued psychiatric patients, since the UK has relegated “CFS” patients to psychiatric care and not traditional medical practices.

“Little is known about the prevalence of XMRV world-wide, much less the incidence of XMRV in ME/CFS or prostate cancer” emphasizes Dr. Judy Mikovits. “WPI and its NCI collaborators are actively engaged with international research teams to investigate these important questions.”

WPI does not recommend the use of anti-retroviral drugs that have yet to be proven to be effective in treating XMRV infection. However, several large pharmaceutical companies have expressed interest in developing anti-retroviral and immune modulating drugs that will effectively treat XMRV associated diseases.

WPI looks forward to the results of other scientific groups around the world, serious about replicating its scientific results, by using the same techniques as WPI and its collaborators. The fact that XMRV was detected in 67 percent of the CFS samples in the U.S. study determined a significant association between XMRV and CFS, demanding a much more serious inquiry by responsible health agencies around the world as to the cause of this debilitating disease.

rr

There was also this response to the UK study by the CFIDS Assoc of America_______

A study testing for evidence of XMRV infection in CFS patients in the United Kingdom has reported negative results. This is the first publication following the article in the top-ranked journal Science from researchers at the Whittemore Peterson Institute, the National Cancer Institute and Cleveland Clinic that garnered worldwide attention from the media and scientific community. The new report, published Jan. 6, 2010, in the open access online journal PLoS ONE, failed to detect XMRV in CFS, but should not be considered a valid attempt to replicate the findings described by Lombardi et al., in the Oct. 8, 2009 Science article.

The PLoS ONE paper by Otto Erlwein, Steve Kaye, Myra O. McClure, Jonathan Weber, Gillian Wills, David Collier, Simon Wessely and Anthony Cleare is titled, “Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome.” The investigators tested peripheral blood DNA from 186 routine clinic attendees who met 1994 (Fukuda) CFS case definition criteria and were well-characterized from participation in prior neuroendocrine and cognitive behavioral therapy studies. These 186 CFS patients were reported to be unwell for a median of four years with high levels of fatigue and disability.

This team of researchers used a special type of DNA “xeroxing” called nested polymerase chain reaction (PCR) reaction to amplify specific segments of the XMRV proviral DNA from the genomic DNA obtained from these 186 CFS subjects. In essence, they were looking to see if XMRV genetic material had integrated into human genetic material, which is a key characteristic of retroviral infection. The experiment included positive, negative and contamination controls, but did not test any samples taken from healthy subjects. The samples were coded so that the origin of the DNA was not known to the person conducting the PCR assays. XMRV was not detected in any of the 186 samples.

Can this study be considered comparable to the results published by Lombardi et al., in Science? In short, no. Both studies included CFS patients defined by the 1994 case definition criteria, but this is where the comparability ends. Here are some of the ways the PLoS ONE and Science methods differ:

* The blood was collected from CFS patients in different types of blood collection tubes. * The genomic DNA was extracted and purified using different techniques. * The amount of genomic DNA included in the amplification assay was different. * Different primer sequences were used that amplified different regions of the XMRV proviral DNA. * The conditions of the PCR amplification assay were different – from the numbers of cycles, to the type of polymerase used.

Should these differences affect an investigator’s ability to detect XMRV? To a microbiologist with experience handling samples and studying various infectious agents (as I am), these variances in procedure could make the difference between detecting XMRV or not.

It very well could be true that XMRV is not present in the U.K. as Erlwein, et al. suggest in their discussion, but it is also possible that the technique used in the PLoS ONE paper was suboptimal due to the different methods employed, when compared to the original experiments conducted by Lombardi, et al.

The U.S. Department of Health and Human Services Blood XMRV Scientific Research Working Group has been established to delineate the research studies that should be undertaken to evaluate whether XMRV represents a risk to the safety of the blood supply. As a first step in this evaluation, analytical panels are being developed by the National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study-II (REDS-II) that will allow for multiple laboratories to standardize methods to optimize sensitive detection of XMRV proviral DNA and viral RNA. Once methods are standardized, these same laboratories plan to test coded panels of blood samples obtained primarily from healthy blood donors and from CFS patients who have been reported to be positive for XMRV.

We look forward to the results of this study and urge that it be completed expeditiously, especially in light of this report from the U.K. In the meantime, be prepared to read about more studies with conflicting findings. Rather than simply accept or dismiss new information, we will help make sense of why discrepant results occur.

Perhaps the most important statement in the PLoS ONE paper is the acknowledgement by this group of investigators that CFS is an incapacitating organic disease affecting millions of people worldwide. Once XMRV detection methods are optimized and made widely available, we encourage this group of researchers to take another look at XMRV as a possible explanation for the organic basis of CFS in the U.K.

Suzanne D. Vernon, PhD, earned her doctorate in virology at the University of Wisconsin at Madison and worked in public health research on infectious diseases at the U.S. Centers for Disease Control and Prevention for 17 years before joining the CFIDS Association of America’s staff as scientific director in 2007. She has more than 70 peer-reviewed scientific publications on topics including human immunodeficiency virus, human papillomavirus, cervical cancer and chronic fatigue syndrome. Dr. Vernon has initiated and participated in numerous international and multidisciplinary research collaborations and she now leads the CFIDS Association’s research program. The CFIDS Association of America is the nation’s largest philanthropic supporters of CFS research.

Petra

Hi Dr. Racaniello. Appreciate your continuing coverage of XMRV. The two groups of scientists who produced these conflicting studies certainly are each standing by their work, as discussed in an article in The Economist: http://www.economist.com/sciencetechnology/disp…

I look forward to watching the truth be hashed out. One quibble with your post – I don't think many CFS sufferers 'believe' XMRV is the etiological agent of the disease. I think a more apt word is 'hope'. I for one, having a caught a 'flu' that hung on for weeks, then months, and now six years, share that hope.

Joan

Unfortunately, “confirmed” CFS patients in the UK are usually anything but. The criteria for CFS depends on having no sign of organic illness, including the signs that are actually hallmarks of CFS.

MEO

We've all seen, and many have personally experienced, conflicting results in science. This is not a he-said she-said issue and the facts will work themselves out in due course. I hope, however, that we manage to extract some important public policy observations from this too. Chronic fatigue syndrome is a chronic disease affecting millions in the US and far more people world wide. Until recently we have not taken many of the needed steps to advance our understanding and expand our treatment options for sufferers from this syndrome, which many clinicians and centers in the US address, well or not, depending on your opinion. Post-infectious fatigue is also an emerging area of concern and is broader than the simple CFS context. Rather than excoriating CDC for their real or perceived shortcomings, let's try instead to provide constructive feedback. Chronic fatigue syndrome is a fine test case for a disease syndrome which costs the US health care system billions of dollars, which occupies the time of frustrated physicians, discourages patients, and in the aggregate does not result in sufficient progress. We can do better. CDC and NIH should be looking at this from these overarching policy perspectives and trying to improve the response of our health care system, including research. Insurers also have a big interest in this and can make another great case for the value of an electronic medical record facilitating our use of epidemiology, clinical findings, and the outcome of interventions.

photochick1

Vincent, thank you for writing about CFS and X MRV. This is an important topic for many people out there.

One thing you need to know about the UK study is it has been initiated by a psychiatrist that believes that CFS is a psychiatric disease. Their samples are certainly not coming from CFS patients but likely more chronically fatigued patients and it is very different from the Lombardi and Al study where CFS patients have been screened carefully.

Also, don't you find it alarming that 0 of their 168 subjects tested negative? Could it be a technical error? Did they look at the rigt place? This they use the right primers? (I am not a scientist, so I will leave it as that)

Lombardi and Al got published after months of peer review, and got published in the most reputable journal in the world. The study has been verified by the Cleaveland clinic and the National institute of Cancer. The UK study got a mere 3 days of peer review and they PAID the journal to get published.

Those are all red flags that shows that maybe this replication study is not valid.

The patients with CFS around the world deserve to be validated for this is a serious disease unfortunately aflicted with a STUPID, NON-SCIENTIFIC name, and has been the object of ridicule for 30 years. Many patients have been bed ridden for 20-30 years, litterally disappeared from the society. Sadly, one the #1 cause of death for CFS patients is SUICIDE, because of the severe stigma nad lack of understanding from family, doctors, disability and health insurance.

Patients deserve some accurate replication of the WPI study. Patients deserve funding for research that will not fall into the hands of psychiatrists like Wessley and Bill Reeves at the CDC who have vested interest that no biological findings turn out to be true.

Thank you for the opportunity to discuss this very important subject and I am looking forward to the next replication study. Please keep an eye for it too!!!

Kati

sciguy

Just to play Devil's Advocate for a second here: those bands in the positive control look pretty weak. If that is the best that the positive control does, I don't think you could put much weight in the negative result. A plasmid-based target for PCR should be off the charts — not just barely detectable.

One nice control here would be the demonstration that they could detect XMRV from an infected mouse using their primers and cycling conditions. I'd bet $5 they can't. Of course, this still doesn't mean that they are wrong…

John

I'm curious and thought maybe since this is a virology blog someone here can help me out.

I've read about how retroviruses are RNA which hijack the body to start producing DNA, with regular viruses being DNA and which do the opposite by producing RNA, but my question is this- what medium encodes the illness beliefs that arise as a result of XMRV retroviral infection? How do so many illness beliefs fit into such a tiny package? Have scientists tried altering the DNA or RNA of the virus in order to short-circuit the illness beliefs contained within? Is there an illness belief receptor that could be blocked somehow? So many questions, so few answers. Any help would be appreciated.

Hip

One of the co-authors of this study, Simon Wessely, has a vested interest in the outcome of such virus studies. Simon has staked his entire academic career on the idea that neurological diseases like chronic fatigue syndrome are caused by purely psychological factors.

Therefore Simon Wessely would be the last person you would want in a study like this: if he finds the virus in CFS patients, he shoots himself in the foot, and his career may never recover. So I wonder just how hard he looked for this XMRV virus.

The odd thing is, the authors declared in this study that they have no competing interests: not true! It is very much in the interest of Simon Wessely to not find this XMRV virus.

THEY ARE NOT STUDYING CFC PATIENTS

Simon Wessely and company often use a different set of criteria to select the patients for their “CFS” studies (such as the Oxford Criteria). These selection criteria are set up so as to include lots of people that are just depressed, and do not have CFS at all.

As a consequence, it is not surprising many studies, based on the Oxford Criteria or similar, find that patients do not have XMRV, or can be cured by antidepressants: they studied the wrong people.

In other words, when Simon Wessely says “CFS”, he actually is talking about “depression”. Wessely loves to play language games, and frequently bends the definition of terms.

The original XMRV research at the Whittemore Peterson Institute used the Canadian Consensus definition of CFS/ME. The advantage of these inclusion criteria is that they actually do select CFS patients!!

SFPam

Dear Most Wonderful Virology Dudes,

Are these two conficting studies actually of equal merit, scientifically-speaking? They are being reported as if they are, and this seems dangerously irresponsible, given the history of weak, misleading research and waylaid research funding in the cfs field. Can you help me understand?

Normally I wouldn’t post this type of information on this type of blog, but I believe it is context that it is as germane to this particular discussion as contaminated labs. It is also a back story that is rarely told and/or possibly acknowledged. Citations are listed at the end.

Factual Background: Dr. Simon Wessely is considered a neo-Freudian. He believes that CFS, along with a number of other syndromes and diseases, are what he terms neurasthenia. Neurasthenia is an archaic theory used prior to the discovery of viruses, microbes and toxins (germ theory model). Adherents believed that diseases had social rather than infectious roots. Hysteria (neurasthenia) was considered to be caused by modern life – including the education of women and spread by the news. The cure was purportedly rest. Somewhere around the 1920s Dr. Freud abandoned the hypothesis of neurasthenia and the focus regarding myalgic encephalomyelitis (a possible subgroup of CFS) shifted to the germ theory model.

Dr. Wessely however, has repeatedly stated that he believes CFS, myalgic encephalomyelitis, and GWS for example are caused by modern fears of viruses, toxins, and microbes not the actual viruses, toxins, or microbes themselves. At the time, he started doing “research” his chosen field of psychosomatic medicine was in the proverbial toilet at least according to Dr. Stanford Friedman then president of the American Psychosomatic Society. Dr Wessely, along with among others Dr. William C. Reeves (junior not senior) of the CDC and UK psychiatric liaison Dr. Peter White, a Wessely and Reeves colleague, are also very strongly influenced by their interpretation of psychiatrist George Engel’s 1977 biopsychosocial theory.

Another theory Dr. Wessely and fellow colleagues have advanced is that of functional somatic syndromes they consider a form of psychosomatic disorder – although there is no official entry for FSS in the DSM. According to this theory, there is no clear-cut biomedical mechanism to explain patient “distress” – or at least none that adherents are willing to acknowledge. In other words, disability and distress and possibly even physiological dysfunction yes, but no pathological changes or abnormalities in the patient. (This may be why Erlwein et al did not note the RNaseL L immunological abnormalities that linked CFS patients in the Lombardi et al study and subgroups of prostate cancer patients who were also found to have XMRV infection. Or why they did not note whether their patient group also met the 2003 Canadian Consensus Definition by Caruthers et al as did CFS patients in Lombardi et al).

(Before I go any further it should probably noted that the World Health Organization’s ICD-10 which UK practitioners are required to abide by, lists myalgic encephalomyelitis exclusively under other organic brain diseases (G93.3). CFS and Post Viral Fatigue Syndrome were added to the tabular listing in 1992. They cannot be moved or concurrently listed under F48 Neurosis category)

Another theory that has been advanced by psychiatrists is that such patients may have a brief, acute organic illness triggered by a virus or other microbe or toxin from which they recover physically, but continue to believe themselves “unwell.” This is called “illness behavior” by psychiatrists. Other CFS experts have repeatedly associated CFS with both a viral trigger and viral persistence in patients for nearly 30 years, but funding has been limited. Is it is possible that institutional bias and bias in the peer review system are also part of the mix? It’s happened before.

Is Dr. Wessely and colleagues with similar views, correct? The honest answer is no one knows. He can’t prove his theories.

Although Dr. Wessely tends to cast patients acting as citizen journalists as the villains, his theories are also very controversial with his peers as is the failure of the CDC’s infectious disease unit, which houses the CFS research program, to do extensive viral studies or track deaths in patients with CFS both of which fall under their purview.

And as is the case with nearly all psychosocial theory there is no objective means of establishing causation although biological psychiatrists (neuroscientists) are looking at many possibilities. As well, in general, there are no definitive biomarkers – psychiatric diagnoses are exclusionary and often become a matter of interpretation. Many believe societal stigma, not science, was one of the reasons homosexuality was originally listed as a psychiatric disorder in the earlier versions of the DSM.

Should Dr. Wessely’s oft stated viewpoints be taken into account when looking at the overall context of this study? Probably. Bias is as common as possible lab contamination and does include ideological bias as well as confirmation bias. Note: Although the above forms of conflict of interest are theoretically acknowledged in the world of science, most journals only require conflicts of interest such as financial involvement of pharmaceutical companies be noted. (Conflicts of interest regarding funding by equally big business interests such as insurance companies are also rarely listed.)

Science has always been a one step forward two steps back and sideways process even when ideological differences are not accounted for. As Vince notes this is one study. I tend to stick with the highly regarded Dr. Silverman whose work on XMRV in prostate cancer patients took three years and who was part of the rigorously peer reviewed Lombardi et al study. He has openly cautioned against hastily produced results regardless of what those results may be. Until variables such as protocols are standardized he is probably correct. Keep an eye peeled for the work of HHS blood safety expert Dr. Jerry Holmberg.

What is perhaps more curious than whether XMRV is found in CFS patients, as another nonpatient science blogger noted, is why many researchers and doctors refuse to acknowledge diseases such as CFS as organic diseases under the category of “we’ll get back to you when we know more.” This is commonly done with multitudes of other diseases for which there are no known biomarkers or etiological explanations including Alzheimer's, Parkinson's and diabetes for example.

I would suspect the answer is multi-factorial. But before thy knee jerks (and no I'm not talking clonus) consider the following:

The most obvious possibility that occurs to me is the substitution of confirmation bias for actual scholarship and intellectual curiosity. For example for years the theory that ulcers were caused by stress instead of bacterium even when the biomedical research was in place fell under the “everybody knows” category – partly because no one was willing to think outside of the confirmation bias box or maybe because one their mind was made up it was set in stone. Or query Ignaz Semmelweis. There are many versions of that fascinating tale.

Another possibility could be stigma and marginalization – as any former member of the AIDS ACT UP group can tell you, stigma and marginalization are very powerful and damaging weapons.

Another could be technology. Multiple sclerosis was commonly referred to by psychiatrists and even medical practitioners as “faker’s disease” until the MRI was invented and demyelination objectively proven.

History – it was also once theorized that patients with Parkinson’s Disease shook because of a frustrated wish to masturbate although that idea fell out of vogue about 70 years ago. Humans have also thought disease to be caused by “bad air” and witchcraft.

Sorry KTSword but I don't agree with your premise. You state that “this is commonly done with multitudes of other diseases for which there are no known biomarkers or etiological explanations including Alzheimer's, Parkinson's and diabetes for example”. I am unaware of any researchers/doctors that refuse to acknowledge these diseases as organic. For example, there is a 25 year history of transplanting fetal tissue in Parkinson's patients to repair the damage to midbrain dopamine neurons — the primary population of cells that degenerate in Parkinson's. And I am pretty sure that the MRI didn't need to be invented to see the damage in MS: one only needs to do an autopsy. Some quick Googling found that EAE was described in 1935, and that white blood cells were known to attack myelin by 1965. The same is true of Alzheimer's and diabetes — no mainstream medical researchers believe that this is anything other than organic, and in each case decades of research has gone into understanding such organic dysfunction.

Anyway, we're interested in whether XMRV really is correlated with CFS. I don't think anyone here is doubting CFS, but there are obviously complications whenever one tries to correlate syndromes without biomarkers to the presence of viruses. Virologists are ultimately interested in discovering causation, and that is much more difficult when you can't easily and concretely define the disease state. The syndrome could be two distinct diseases. Or twenty three. Having a defined biomarker permits a more objective measure of correlation, but has nothing to do with believing a patient.

Personal attacks on leading professionals in the domain occur with regrettable frequency, but they have no place in the Scientific Method and review processes. Any criticism should be evidence based or identify flaws in the process.

In this case the paper identifies the population that the study was drawn from as “The study recruited 186 patients … had undergone medical screening to exclude detectable organic illness, … were routine clinic attendees … who had taken part in prior studies of neuroendocrine functioning and/or of CBT.” The clinic is in the heart of London (SE5) and getting there involves an arduous journey (when many chronic CFS sufferers struggle to leave their house). Many UK CFS patient groups boycott this clinic which only offers psychiatric / CBT / GET therapies, as they believe this illness to be of biological origin. This is “clinic participants filter” therefore tends to exclude the very class of CFS patient who might have XMRV, yet this bias has been ignored in the study design.

This study only tests attendees at Prof Wessely's clinic. Extending its conclusions to the entire UK CFS community is a step too far.

KTSword

The nice thing about discussion is that everyone has a contributing point of view. If you read closely, there is no personal attack or opinion here. Just background information with citations. I do apologize if it struck anyone as anything other than that.

Scientists above all should question all assumptions not just the ones that fit their personal view of the world. Impossible to do evidence based science without doing that first. Good points.

Anyone searching for more information about conflict of interest and competing interests can find additional information on the website of the World Association of Medical Editors.

Thanks sciguy. I like many of the points you made. Just for clarification the neuroscientist whose statement I paraphrased didn't say scientists and clinicians don't believe Parkinsons, diabetes or Alzheimer's are organic although many did at one time – it's all there in medical history. His question was why some don't categorize CFS in that same category. Notice he did not say all, he said some. Sorry if that wasn't clear.

Of course the signs and symptoms of MS are there just as the signs and symptoms of CFS are present, but that doesn't change history. But, as you have appropriately pointed out, biomarkers are different than belief and interpretation.

I read and research widely, both in the lay press and in the scientific world of research as well as talking to everyone from scientists to patients in many fields – that's my job. I fully understand the scientific process and find the twists and turns fascinating.

Based on what I've come across, and I can only speak to my own experience, many people are unaware of the biomedical research that has been done in the field of CFS and yes, some do publicly doubt the organic nature of CFS.

Interestingly enough, a number of biomarkers have been found in CFS that require further study in well characterized patient sets and controls. For example, neurologist and research Ben Natelson used protemics to find biomarkers in the spinal fluid of CFS patients back in '05 I believe, but couldn't obtain further funding. Neurologist Frank Duffy did a study involving hundreds of patients and controls and was able to fully differentiate between CFS patients and depressed patients and controls using qEEG technology. But it takes funding for further study regardless of what is being studied, the variables being questioned regardless of the eventual outcome. That is a reality all scientists live with. I'd wave my magic wand and provide funding for all if the darn thing wasn't broken and the warranty expired.

As noted at the top of the post the point was to stimulate discussion only. I simply referred to a comment made by another scientist on his blog. Playing devil's advocate doesn't mean that is what I personally believe or disbelieve – it is nothing more than suggesting and then questioning variables. As a sciguy I'm sure you do that all the time as you showed from your post. Cool.

You are right, scientists don't know if they are dealing with a discrete entity or many organic diseases with a common core. Dharam Ablashi, who co-discovered HHV-6, for example theorizes that the signs and symptoms of the patients are dependent on what type of latent viruses are triggered by something such as for instance a retrovirus. I think others would hypothesize that exposure to toxins could also trigger such a cascade, but don't quote me on that.

Others, such as Vince, believe there has to be something else in play or else everyone who tests positive for XMRV would have CFS or in the case of men, prostate cancer – it's a guy thing. Robert Silverman at the Cleveland Clinic links a defect in the RNaseL pathway between the tested subgroup of prostate cancer patients and the WPI, NCI and Cleveland Clinic study on XMRV in CFS patients. UK Microbiology professor Jonathan Kerr is researching the genetics angle regarding viruses and CFS and his work too is interesting. He is also doing research on XMRV. Others are looking into a possible link with XMRV and GWS. It will interesting to see where this all goes.

(PS I'm a medical journalist – neither patient nor scientist – so my professional training is to question everything with great glee I might add ;D. My personal point of view is irrelevant and unstated as is consistent with my profession.)

And yes, association is not causation. Did you read Mark Crislip's most recent post on Hill's criteria? Interesting read.

Terry Ellison

My point comment about personal attack related to an earlier post in this thread. However this post was about a report of an XMRV study, and I am not sure what a critique of Wessely et al got to do with the finding expressed in the paper and the work of Dr Erlwein1, Professor McClure et al? I happen to agree broadly with your POV, but again I don't think that this is relevant here.

However, I think that my point that the selection of the cohort from patients attending KCH CFS clinic has introduced a bias ,which is a flaw in the trial design, IS relevant to this paper.

Hip

Terry Ellison: Unfortunately, you seem unaware of the history of Simon Wessely, and his realpolitikal influence. It is Simon Wessely and his psychiatric network of friends – often referred to as the “Wessely School” (Google this) – that has significantly influenced government policy towards CFS/ME patients. This not just pure, innocent science as you like to believe.

It is Wessely School influence over the last decade or two that swayed the Centers for Disease Control away from biomedical and infectious disease research model of CFS/ME, and into his odd territory of the purely psychological explanations for CFS/ME, with no interest on how this affects the CFS patients.

Listening to Wessely's ideas, it feels as if the Scientific Enlightenment never happened, and we were still in the Middle Ages, believing that spells and sorcery as the explanation of illnesses.

Simon Wessely always seeks to impose his odd views on CFS on government institutions, and has regularly succeeded in shaping CFS policy at the CDC and the UK National Health Service by his astute political abilities.

Wessely does not answer to, or represent, the views of the CFS/ME patient community; rather, he is a self-appointed autocrat in the domain of government CFS policy, both in the UK, and the US.

Petra

Hi KTSword. I've really enjoyed your comments here. It's great to hear from folks who are knowledgeable but are not patients, doctors, or directly-involved scientists. I think you've hit the nail on the head, that what's truly momentous about the XMRV story is the fact that yet another a disease once thought to be psychosomatic is being proven to be organic. I hope you're writing up an article on the topic – let us know when we can read it!

KTSword

Terry,

Perhaps if I explain it differently it will make more sense to you – or not.

For example, let's say a hypothetical group of scientists found that a specific drug had an “unacceptable level” of deaths associated with it and another paper immediately came out saying that's not what they found. If let's say some of the authors of the second paper were being paid by the manufacturer of the drug in question many might consider that a competing interest and one they would want to know about. It wouldn't automatically mean that their research wasn't accurate, but it would be considered germane.

Ideology is also considered a competing interest in the world of science. Scientists who have spent a long career positing a hypothesis at direct odds with an earlier study may unconsciously or not so subconsciously have findings that preserve their stance. This is called confirmation bias. That is reality that is backed up by more research than I care to get into because that is a tangent.

Does it automatically make it true? No. Nor if you go back and re-read my post did I say that. I simply gave information backed up by citations – what other people do with it is not under my control nor do I wish it to be. It is what is known as context. Facts rarely exist in a vacuum. Probably enough said on that subject.

I think you have a good point worthy of consideration re: the cohort, but then I've made that point also so perhaps I have a bit of confirmation bias of my own.

Thanks for the discussion.

Hip

Likewise, I also enjoyed the writing of KTSword; it was interesting to get more info on the background and interests of Simon Wessely.

But changing the subject to focus on a broader picture:

What must be realized here is that CFS connection to viral causes is not an isolated case, but rather forms part of a bigger picture, in which a whole array of common diseases (of hitherto unknown etiology) are now being linked to, and perhaps completely caused by, viral, bacterial, and other pathogens. If you start to look at diseases, from the neurological ones like MS and Alzheimer's, to endocrine disease like diabetes, and the various type of cancer; well, these are all recently being linked to microbial pathogens.

One book that covers this subject, and is well worth reading, is “Plague Time: How Stealth Infections Cause Cancer, Heart Disease, and Other Deadly Ailments”, by PAUL W. EWALD ( http://www.edge.org/q2006/q06_12.html ).

So this current era in medical research is a watershed in thinking: in the future, we will realized the the pathogens we harbor in our body can and frequently do instigate serious disease.

I believe we come to appreciate that the human body is far more robust than we currently think. We are accustomed to disease being a regular part of human life, and assume disease is the result of a sort of wear and tear process on the body; or perhaps we think that disease must result from bad genes.

These explanations are involved in creating diseases as well, but for the majority of diseases, it will likely turn out, I think, that the various microbes that live in our body are their trues cause. Think of this a very good news: it means if we can devise means to eliminate these microbes, out bodies will enjoy near-perfect health.

Thus far more research into the life of the various categories of microbes is necessary, in or to take us into the Garden of Eden of superior health.

Terry Ellison

I agree the given SW and AC's previous academic output and public position, then their adopting a positive position on would be a bit like “turkeys voting for Thanksgiving” as our US colleagues might say. But all that the KCH team did — if I read the paper correctly — was to identify the patient cohort and supply the samples. Prof MM's team did the virology, and they've got a pretty good track record in retrovirus work. One easy way to resolve this would be for the Imperial team to do a repeat using a patient cohort drawn from the Myhill Mitochondrial Dysfunction study patient group (PMID: 19436827).

sciguy

OK, then I think we are on the same page. I was simply responding to what you said in the post regarding the other diseases – I would agree that some do doubt the organic nature of CFS. And I am all for stimulating discussion! I'd like to point out for others reading this (see Petra below) that I am not a directly-involved scientist: I earned my Ph.D. in virology but moved on to stem cell research. I now run a lab focused on human pluripotent stem cells, so I don't have an iron in this fire either way. I was only trying to interpret the data present (or not present as the case may be) on the gel. I would consider this a hobby of sorts — a luxury provided by Dr. Racaniello (Thanks, Vince, you're the man!) As a scientist, I enjoy interpreting and debating data.

In fact, just to play Devil's Advocate again, I'd respectfully disagree with Vince that something needs to be in play other than XMRV. Maybe some people are challenged with a larger infectious dose, or are inoculated via different routes, have different genetics/immune systems that cause different outcomes, etc. HSV1 almost never infects the CNS luckily for both virus and host. But every once in a while, there is an unfortunate event that causes it to run rampant and destroy portions of the brain. Could there be another trigger/virus collaborating in this event? Maybe, but to my knowledge there's been no evidence of this. I think that there are many examples of common infections that rarely cause complications — this could be similar.

In any case, don't take it personally! Your training, like mine, is “to question everything with great glee”. Just trying to hold your feet to the fire. Feel free to do the same to me : )

MEO

Two questions offered for comment:

1. In the last 20+ years we've seen several prominent new viral syndromes emerge. Has expert panel articulated a general scientific strategy for investigating the identity of a putative viral etiology? (A question well put to CDC and NIH, who may have to do this work in any event, and not too removed from the kind of pandemic response planning they have been undertaking recently.)

2. Those of us who are scientists, certainly from my generation, often view scientific debates in the lay press as somewhat talking out of school. We prefer to see these matters worked out in professional journals by “the experts”. But, the changing environment of communications seems to be taking these subjects more and more to the popular press and forcing the debate to be in the media – a la global warming. In reporting such stories, it is difficult to see how to walk the line between simply and dispassionately “informing” the public and naively providing misinformation. Here, for instance, we have an emotional and time-sensitive drama involving people's lives and health, one in which the public has every right to demand urgency and progress from its public health system. I may be acquiesing, reluctantly, to the view that studies, like the one from the UK, following so quickly on the heels of the initial Science paper, are as much a press release as a scholarly publication. If this is going to be the venue, then, maybe, what we need is exactly the opposite of what I grew up with, that is, very explicit and vigorous public critique of such papers on whatever grounds seem relevant?

asus389

I am not at all certain that “there has to be something else going on here or else everyone with XMRV would get CFS”. Perhaps people who tested positive for XMRV are at different stages of infection? Perhaps XMRV infection, like HTLV infection doesn't always manifest itself as disease? Or perhaps XMRV, like FeLV (a very close cousin) both causes different symptoms depending on stage of infection and also doesn't progress to every stage in everyone who is exposed. FeLV is fought off completley by a decent amount of the cats exposed, in some they become infected but its dormant and doesn't cause illness, some it causes persistent viremia and immune supression, some cats get cancer, etc… I guess my point is, we shouldn't just assume its a simple all or nothing illness. Even people with HIV don't manifest symptoms sometimes for years… and some people exposed to HIV never become positive – it can take many exposures to become infected in some cases.

asus389

I'll add another thing that isn't being accounted for here – the idea that all illnesses can be tested for by a blood test. What if XMRV is only in the blood during a certain phase of the illness? Its possible then that testing blood for it isn't going to show 100% postivity in everyone with symptoms all the time. Blood is a great place to start looking for something, but not all pathogens primarily infect or are in the blood compartment all or any of the time.

Hip

The general problem in translating scientific facts, theories, assumptions, hypotheses and contingencies for the consumption and understanding of the general public is that that the lay public are generally not happy to deal with the inherent levels of uncertainly that are normally found in science. The lay public are often just looking for hard public facts, and are not conformable with conditionals – with the truth of any fact being contingent upon various others circumstances. Dealing with this delicate fabric of logical interrelation is what scientists enjoy and excel at, but the public generally just want a simple answer. This is the most difficult problem in disseminating science into the public arena.

However, the great advantage about this increased public involvement, via the Internet, etc, is that it, in a sense, brings the ideals of democracy into even the tiniest microcosms of human activity. The case in point here is the highly controversial story of Wessely School ideas of CFS dominating the official government agendas in CFS/ME. The autocratic control that the Wessely School has taken in the microcosm of CFS/ME is utterly undemocratic, and the general noise and hubbub that CFS patients are making about this indicates that it is very unpopular in this community. This is no different to peaceful marches in the streets to indicate dissatisfaction with the government.

So this public involvement in such microcosms is ultimately a very good development in human affairs. The only element that is missing is the mechanism – the equivalent of voting – to instal into power the scientists of choice, and the remove from “office” those people who have created the kind of mass dissatisfaction we see here in the CFS/ME community.

This not to say that Simon Wessely should not be allowed to continue with his academic interests; it is just to say that should not longer be given the reigns of power and the autocratic control of setting government policy and research strategy on CFS/ME, when there is such dissent amongst the CFS/ME community.

Of course, one would expect the Wessely School to collectively respond: “let them eat cake!”, in their obliviousness to the suffering they have created.

But the question is here, how do you turn Internet dissent into action to instigate change?

Hip

On the subject of something in addition to XMRV being at play inCFS/ME:

What the WPI are saying is that it is still believed that CFS/ME is caused by a low-level chronic infection of the same viruses that were originally suspected as the main agents in CFS/ME (viruses such as enterovirus, Epstein-Barr virus, HHV-6, parvovirus B19, cytomegalovirus), but that having XMRV explains why, in these people, these viruses are not so well controlled by the immune system, and therefore go on to create a systemic infection – the infection that is behind all the observed bodily-wide odd symptoms of CFS/ME, from periodontal decay to arthralgias.

However, I would wager – if I had to speculate – that it may be only be the more severe ME patients that will test positive for XMRV, simply because viruses like enterovirus can be pretty nasty just on their own. Chronic enterovirus infections are already implicated as the cause of various other diseases, such as diabetes, heart attacks, and heart atrophies, arthritis, ADHD, Guillain-Barre syndrome, and many others. Since these diseases may be caused by chronic enterovirus, it is clear that once in the body, some of the nastier enteroviruses can manage quite well on their own, thank you very much, in precipitating chronic disease, without the immunosuppressive assistance XMRV.

So while the XMRV is probably going to be a major part of the explanation of the more serious ME, we should lose site of the the fact that enterovirus, Epstein-Barr, etc may still have the ability to precipitate milder CFS/ME (and many other diseases) on their own.

http://www.virology.ws profvrr

There are problems with both studies that won't be resolved by the parties arguing with one another. The best way to resolve the differences is for the two groups to exchange blood samples and run their PCR assays. But this will never happen, because both parties have big egos and appear to be more concerned with their side of the story than in answering the question. Science should be about helping people have healthy lives, not having more publications or grant money than the other group. Such situations underscore one of the worst aspects of science (the worst being fraud), and in part lead to public distrust of scientists.

KTSword

I just love warm tootsies in the winter

Gertrude

I read an article and a comment on this debate this morning that pretty well confirm to me that the Imperial College cannot be trusted to give accurate testimony about their results and have already knowingly lied in their presentations to the press.

Professor Myra McClure, from Imperial College London, “…. If it had been there, we would have found it. The lab in which we carried out the analysis had never housed any of the murine (mouse/rat) leukaemia viruses related to XMRV, and we took great care to ensure there was no contamination. We are confident that our results show there is no link between XMRV and Chronic Fatigue Syndrome, at least in the UK.”“McClure implies in her statement that the positive test results in the US were due to contamination. There are several facts she must have been aware of when making her statement that stongly suggest this to be untrue.The first is that there were 2 groups in the WPI study. The first group of 100 CFS patients had a XMRV +ve by PCR rate of 67% . The second group of 208 healthy controls had a XMRV positive rate of 3.7 %. Had the WPI results been positive because of Laboratory contamination there would have been more even numbers in both groups but indeed the differences in the 2 groups were marked.Any scientist or technician would know this. Please correct me if I'm wrong about this. If Im correct then that means Myra McClure is telling a deliberate lie by implication and that puts the intent and results of the Imperial College study itself into question. She is using false implication to bolster acceptance of her results by people who dont have a scientific backgound.

The results are the strongest ever reported linking a virus to a specific illness.

The WPI samples were also tested in 3 laboratories, including that of Dr Silverman who co -discovered this virus 3 years ago in research unrelated to CFS. So the imperial College are saying they are sure the discoverers of the virus cannot test for the the virus they discovered. Myra must have heard by this stage that WPI is testing 500 UK samples with the % ages are coming out the same as the WPI study so that blows the idea that its not in the UK.

GrantH-C

There have been numerous studies that suggest that patients with CFS are chronically immunosuppressed. This phenomenon is also seen in patients with depression. It is therefore not surprising that a higher incidence of virus is detected in an immunosuppressed group versus controls. Such studies are likely to throw up a number of “possible aetiologic viruses” due to the higher incidence of infection in CFS patients. The differences in the USA and UK studies may simply reflect the difference in circulating virus populations – if there is less XMRV in the UK then fewer patients will be infected due to their immunosuppressed state. Too many people are taking about XMRV being an aetiologic agent with minimal fulfillment of Koch's postulates.

Gertrude

Grant

The proof is found when you eliminate the virus and the symptoms disapear. There have been instances where CFS patients undergoing B cell depletion therapy for cancer have had dramtic improvements in their CFS symptoms.

WPI culture this virus from CFS patients blood and have found the virus in patients B cells. B cell depletion forces the body to make new B cells from stem cells so theoretically these new B cells would be temporarily free of the virus but would at some point become reinfected and relapse would happen. This is exactly what hs happened to these cancer patients. A study is also being done withCFS patients of this therapy and the patients improve then relapse as would be expected to happen. But after the third treatment session are improving more and more.

There could be another reason for this therapy's unexpected results however if XMRV inhabits the B cells and if they use this therapy in conjunction with antiretrovirals they may manage to prevent reinfection of the newly minted B cells and reduce infection level to where patients can get back to their lives. So its possible that one of the most important steps in proving causation is already in testing and getting good results. Ive had this illness for 34 years and have always been a very positive person. I think people who are depressed know it. They don't dispute it and are treated with a lot of success. I am in agonising pain. The psycobabble has only delayed any real research into this condition and eaten up the good years of my life. These type of retroviruses are known to supress the imune system and the rnase l immune abnormality found in cfs was also found in the agreesive prostate cancer patients they found this virus in and thats one of the reasons they tested cfs patients for it.

sciguy

Hear, hear. I suppose another avenue for resolution is by having a number of groups perform their own analysis, something very likely given the importance of finding The Truth.

Petra

@ sciguy: Thanks for telling a bit about your background, and thanks for coming by here to debate/discuss – it's fantastic to hear discussion between two well-informed people who don't have a horse in this race.

Petra

“I may be acquiesing, reluctantly, to the view that studies, like the one from the UK, following so quickly on the heels of the initial Science paper, are as much a press release as a scholarly publication. If this is going to be the venue, then, maybe, what we need is exactly the opposite of what I grew up with, that is, very explicit and vigorous public critique of such papers on whatever grounds seem relevant?”

Very interesting point, MEO!

Petra

Hi GrantH-C. I've had 'the flu' for quite a while now – sore throat, diarrhea, skin rashes, body aches, fatigue. You know how the flu feels? Yeah. I've had it for 6 years. Please, I'm begging you, don't talk like you have the answer and it's depression.

I just thank god that real scientists are working on this (and blogging on it – Thanks Dr. R!). Bring on the controversy, and let's get this damn thing figured out.

KTSword

Just a few remarks re: Koch's Postulates (remarkable man)First, strictly for the record, it should be pointed out that viruses in particular can never fulfill all of Koch's Postulates. Also, Koch's Paradigm, which was first postulated around 1879, assumes that one organism equals one disease. There is considerable evidence for CFS, at least in subgroups, and an increasing number of complex diseases, that there may be more than one pathogenic organism involved. There is a whole lot of water under the scientific bridge since the late 1800s. Please remember as many scientists, including CFS experts who are involved in other XMRV studies, have noted the data on XMRV is preliminary. No matter what the disease. XMRV wasn't even definitively discovered until 2006. And it could be there are others in play that have yet to be identified. Lack of a name or lack of “discovery” doesn't mean a virus or disease doesn't exist – just that humans don't know everything and probably never will. The data associating CFS with several other viruses is not preliminary, but there again science is back to the same old same old – are scientists studying the exact same groups when it comes to CFS? The fewer variables controlled for, no matter what the subject, the more likely it is that specific results cannot be given a free pass when extrapolating them to other groups. If a retrovirus is in the mix it should also be noted that retroviruses in general are notorious for kicking up latent viruses that are normally harmless once the initial infection has run its course. Scientists didn’t know that when Koch was alive. Devanur L.D., Kerr J.R. “Review: Chronic Fatigue Syndrome.” Journal of Clinical Virology 37 (2006) 3:139-150

Also, just a follow-up on the questions regarding whether the UK study studied the exact same group of CFS patients as did the October 2009 study. In short – no. So in essence different protocols, different patients in a different geographical location – different results. UK psychiatrist Anthony Cleare, one of the authors of the UK XMRV study published in PLoS, posted a reply to many of the questions that have been discussed here and elsewhere. The post can be found in the comments section below the original study and was posted on 12 Jan 2010 at 19:55 GMT. As is perhaps to be expected they stand by their results despite the differences. Also perhaps to be expected their citations were nearly all studies in which one of the UK authors participated in. This may reflect a better familiarity with their own research. Or it may reflect a rejection of any studies that do not support their assertions. That sword cuts many ways. To sum up:Unlike the WPI, National Cancer Institute and Cleveland Clinic study on XMRV published in Science back in October, the UK cohort was not defined using the 2003 Canadian Consensus Protocols and while Dr. Cleare suggests that some patients may have met that definition, but they did not select the samples on that basis nor is there a citation to suggest that he knows that for sure. Dr. Cleare also notes that CFS patients with cancer were excluded from the XMRV study. The question is whether the patients had CFS before they were diagnosed with cancer. Many diseases are co-morbid, but it does muddy the water when trying to figure out what causes what particularly when some symptoms are common to both diseases. Although Dr. Cleare cites immunological studies he did not include studies regarding the RNaseL link that led scientists at the WPI, NCI and Cleveland Clinic to find the connection between the prostate cancer patients Dr. Silverman studied and XMRV in CFS patients. This also suggests another variable for which the UK scientists did not control. Something I think may also be pertinent is whether the patients from whom the UK samples were taken meet the 1994 Fukada definition at the current time. It should also be noted that Dr. Cleare states that members of the King’s College Clinic did not chose the samples tested.

anonymous

HI I am from Europe and got cfs there and just got tested positive for XMRV in the US, so it is not a regional thing

There are reports now of people in the UK testing positive for XMRV by culture using the same methods as the Science study through VIP Diagnostics. Some of these folks have never been to the US. As a physician disabled by CFS, I am looking forward to future studies on XMRV and CFS.

It's true that ideally, science should not be contaminated by politics but there's something terribly wrong when some prominent researchers (like the CDC) have not listened to the health concerns of sufferers over the last 3 decades and have not even collaborated with experienced CFS clinicians and researchers.

For example, you will see nowhere on the CDC website that the full recovery rate for CFS is less than 10% (this from several scientific follow-up studies tracking people more than a decade) although people with the illness know this. The impression the CDC site gives is that people are sick for a few years and then recover. Hence, there have been no studies looking at the long-term medical consequences of CFS.

As a second example, the CDC did a population-based study out of Wichita diagnosing people with CFS despite them working a median of 40 hours a week when in other studies, the numbers are much lower – e.g. 9-16% of CFS subjects even working at all, much less full-time. It doesn't sound they are diagnosing people correctly yet this is only recognized as a concern in one paper coming out of the series of papers they published on this group.

As a medical student, I was taught the importance of listening to patients; I would hope the same would apply to medical researchers.

MR

There are reports now of people in the UK testing positive for XMRV by culture using the same methods as the Science study through VIP Diagnostics. Some of these folks have never been to the US. As a physician disabled by CFS, I am looking forward to future studies on XMRV and CFS.

It's true that ideally, science should not be contaminated by politics but there's something terribly wrong when some prominent researchers (like the CDC) have not listened to the health concerns of sufferers over the last 3 decades and have not even collaborated with experienced CFS clinicians and researchers.

For example, you will see nowhere on the CDC website that the full recovery rate for CFS is less than 10% (this from several scientific follow-up studies tracking people more than a decade) although people with the illness know this. The impression the CDC site gives is that people are sick for a few years and then recover. Hence, there have been no studies looking at the long-term medical consequences of CFS.

As a second example, the CDC did a population-based study out of Wichita diagnosing people with CFS despite them working a median of 40 hours a week when in other studies, the numbers are much lower – e.g. 9-16% of CFS subjects even working at all, much less full-time. It doesn't sound they are diagnosing people correctly yet this is only recognized as a concern in one paper coming out of the series of papers they published on this group.

As a medical student, I was taught the importance of listening to patients; I would hope the same would apply to medical researchers.

The reaction products were fractionated by electrophoresis on an agarose gel, and the DNAs were visualized by staining with a dye. As shown in the figure, positive control samples containing XMRV DNA produced DNAs of the expected sizes!

http://www.moldtesting.org Mold Testing Elmira

Great site and I look forward to checking out your updates!

Pat McGinn

Looking at earlier info about CFS/M.E. , there is no virus involved in this disease. The opposite is true: fewer/less/no viral disease but more bacterial infections. The cause is the seaweed food additive, carrageenan, that permanently becomes incorporated into cells and causes huge damage. Researchers have found with lab animals that ingestion of carrageenan causes a large reduction in viral replication by the host. Which fits in with the Akureyri outbreak in Iceland (1947/8) – people with this disease did not get polio, which was spreading thru’ Scandinavia at this time.